Ana I. Manzano

Gravitational and magnetic field variations synergize to cause subtle variations in the global transcriptional state of Arabidopsis in vitro callus cultures

BackgroundBiological systems respond to changes in both the Earths magnetic and gravitational fields, but as experiments in space are expensive and infrequent, Earth-based simulation techniques are required. A high gradient magnetic field can be used to levitate biological material, thereby simulating microgravity and can also create environments with a reduced or an enhanced level of gravity (g), although special attention should be paid to the possible effects of the magnetic field (B) itself.ResultsUsing diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to five environments with different levels of effective gravity and magnetic field strengths. The environments included levitation, i.e. simulated μg* (close to 0 g* at B = 10.1 T), intermediate g* (0.1 g* at B = 14.7 T) and enhanced gravity levels (1.9 g* at B = 14.7 T and 2 g* at B = 10.1 T) plus an internal 1 g* control (B = 16.5 T). The asterisk denotes the presence of the background magnetic field, as opposed to the effective gravity environments in the absence of an applied magnetic field, created using a Random Position Machine (simulated μg) and a Large Diameter Centrifuge (2 g).Microarray analysis indicates that changes in the overall gene expression of cultured cells exposed to these unusual environments barely reach significance using an FDR algorithm. However, it was found that gravitational and magnetic fields produce synergistic variations in the steady state of the transcriptional profile of plants. Transcriptomic results confirm that high gradient magnetic fields (i.e. to create μg* and 2 g* conditions) have a significant effect, mainly on structural, abiotic stress genes and secondary metabolism genes, but these subtle gravitational effects are only observable using clustering methodologies.ConclusionsA detailed microarray dataset analysis, based on clustering of similarly expressed genes (GEDI software), can detect underlying global-scale responses, which cannot be detected by means of individual gene expression techniques using raw or corrected p values (FDR). A subtle, but consistent, genome-scale response to hypogravity environments was found, which was opposite to the response in a hypergravity environment.

Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings

BackgroundCell growth and cell proliferation are intimately linked in the presence of Earth’s gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter.ResultsWe studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells.ConclusionsIn our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.

Effect of magnetically simulated zero-gravity and enhanced gravity on the walk of the common fruitfly

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly (Drosophila melanogaster) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earths surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0g*) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2g*) and a normal gravity environment (1g*) within the spatially varying field. The flies had a larger mean speed in 0g* than in 1g*, and smaller in 2g*. The mean square distance travelled by the flies grew more rapidly with time in 0g* than in 1g*, and slower in 2g*. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

Structural studies of novel glycoconjugates from polymerized allergens (allergoids) and mannans as allergy vaccines.

Immunotherapy for treating IgE-mediated allergies requires high doses of the corresponding allergen. This may result in undesired side effects and, to avoid them, hypoallergenic allergens (allergoids) polymerized with glutaraldehyde are commonly used. Targeting allergoids to dendritic cells to enhance cell uptake may result in a more effective immunotherapy. Allergoids coupled to yeast mannan, as source of polymannoses, would be suitable for this purpose, since mannose-binding receptors are expressed on these cells. Conventional conjugation procedures of mannan to proteins use oxidized mannan to release reactive aldehydes able to bind to free amino groups in the protein; yet, allergoids lack these latter because their previous treatment with glutaraldehyde. The aim of this study was to obtain allergoids conjugated to mannan by an alternative approach based on just glutaraldehyde treatment, taking advantage of the mannoprotein bound to the polymannose backbone. Allergoid-mannan glycoconjugates were produced in a single step by treating with glutaraldehyde a defined mixture of allergens derived from Phleum pratense grass pollen and native mannan (non-oxidized) from Saccharomyces cerevisae. Analytical and structural studies, including 2D-DOSY and 1H-13C HSQC nuclear magnetic resonance spectra, demonstrated the feasibility of such an approach. The glycoconjugates obtained were polymers of high molecular weight showing a higher stability than the native allergen or the conventional allergoid without mannan. The allergoid-mannan glycoconjugates were hypoallergenic as detected by the IgE reactivity with sera from grass allergic patients, even with lower reactivity than conventional allergoid without mannan. Thus, stable hypoallergenic allergoids conjugated to mannan suitable for using in immunotherapy can be achieved using glutaraldehyde. In contrast to mannan oxidation, the glutaraldehyde approach allows to preserve mannoses with their native geometry, which may be functionally important for its receptor-mediated recognition.

Mite allergoids coupled to nonoxidized mannan from Saccharomyces cerevisae efficiently target canine dendritic cells for novel allergy immunotherapy in veterinary medicine

We have recently reported that grass pollen allergoids conjugated with nonoxidized mannan of Saccharomyces cerevisae using glutaraldehyde results in a novel hypoallergenic mannan-allergen complex with improved properties for allergen vaccination. Using this approach, human dendritic cells show a better allergen uptake and cytokine profile production (higher IL-10/IL-4 ratio) for therapeutic purposes. Here we aim to address whether a similar approach can be extended to dogs using canine dendritic cells. Six healthy Spanish Greyhound dogs were used as blood donors to obtain canine dendritic cells (DC) derived from peripheral blood monocytes. Allergens from Dermatophagoides farinae mite were polymerized and conjugated with nonoxidized mannan. Nuclear magnetic resonance (NMR), gel electrophoresis (SDS-PAGE), immunoblotting and IgE-ELISA inhibition studies were conducted to evaluate the main characteristics of the allergoid obtained. Mannan-allergen conjugate and controls were assayed in vitro for canine DC uptake and production of IL-4 and IL-10. The results indicate that the conjugation of D. farinae allergens with nonoxidized mannan was feasible using glutaraldehyde. The resulting product was a polymerized structure showing a high molecular weight as detected by NMR and SDS-PAGE analysis. The mannan-allergen conjugate was hypoallergenic with a reduced reactivity with specific dog IgE. An increase in both allergen uptake and IL-10/IL-4 ratio was obtained when canine DCs were incubated with the mannan-allergen conjugate, as compared with the control allergen preparations (unmodified D. farinae allergens and oxidized mannan-allergen conjugate). We conclude that hypoallergenic D. farinae allergens coupled to nonoxidized mannan is a novel allergen preparation suitable for canine allergy immunotherapy targeting dendritic cells.

Functional alterations of root meristematic cells of Arabidopsis thaliana induced by a simulated microgravity environment

Environmental gravity modulates plant growth and development, and these processes are influenced by the balance between cell proliferation and differentiation in meristems. Meristematic cells are characterized by the coordination between cell proliferation and cell growth, that is, by the accurate regulation of cell cycle progression and the optimal production of biomass for the viability of daughter cells after division. Thus, cell growth is correlated with the rate of ribosome biogenesis and protein synthesis. We investigated the effects of simulated microgravity on cellular functions of the root meristem in a sequential study. Seedlings were grown in a clinostat, a device producing simulated microgravity, for periods between 3 and 10days. In a complementary study, seedlings were grown in a Random Positioning Machine (RPM) and sampled sequentially after similar periods of growth. Under these conditions, the cell proliferation rate and the regulation of cell cycle progression showed significant alterations, accompanied by a reduction of cell growth. However, the overall size of the root meristem did not change. Analysis of cell cycle phases by flow cytometry showed changes in their proportion and duration, and the expression of the cyclin B1 gene, a marker of entry in mitosis, was decreased, indicating altered cell cycle regulation. With respect to cell growth, the rate of ribosome biogenesis was reduced under simulated microgravity, as shown by morphological and morphometric nucleolar changes and variations in the levels of the nucleolar protein nucleolin. Furthermore, in a nucleolin mutant characterized by disorganized nucleolar structure, the microgravity treatment intensified disorganization. These results show that, regardless of the simulated microgravity device used, a great disruption of meristematic competence was the first response to the environmental alteration detected at early developmental stages. However, longer periods of exposure to simulated microgravity do not produce an intensification of the cellular damages or a detectable developmental alteration in seedlings analyzed at further stages of their growth. This suggests that the secondary response to the gravity alteration is a process of adaptation, whose mechanism is still unknown, which eventually results in viable adult plants.

Early Effects of Altered Gravity Environments on Plant Cell Growth and Cell Proliferation: Characterization of Morphofunctional Nucleolar Types in an Arabidopsis Cell Culture System

Changes in the cell growth rate of an in vitro cellular system in Arabidopsis thaliana induced by short exposure to an altered gravity environment have been estimated by a novel approach. The method consisted of defining three structural nucleolar types which are easy and reliable indicators of the ribosome biogenesis activity and, consequently, of protein biosynthesis, a parameter strictly correlated to cell growth in this cellular system. The relative abundance of each nucleolar type was statistically assessed in different conditions of gravity. Samples exposed to simulated microgravity for 200 min showed a significant decrease in nucleolar activity compared to 1g controls, whereas samples exposed to hypergravity (2g) for the same period showed nucleolar activity slightly increased,. These effects could be considered as an early cellular response to the environmental alteration, given the short duration of the treatment. The functional significance of the structural data was validated by a combination of several different well-known parameters, using microscopical, flow cytometry, qPCR and proteomic approaches, which showed that the decreased cell growth rate was decoupled from an increased cell proliferation rate under simulated microgravity, and the opposite trend was observed under hypergravity. Actually, not all parameters tested showed the same quantitative changes, indicating that the response to the environmental alteration is time-dependent. These results are in agreement with previous observations in root meristematic cells and they show the ability of plant cells to produce a response to gravity changes, independently of their integration into plant organs.

Effect of Bisphosphonates on Root Growth and on Chlorophyll Formation in Arabidopsis thaliana Seedlings

Bisphosphonates (BPs) are analogues of pyrophosphate in which the oxygen bridge between the two phosphates is replaced by a methylene group (–CH2–). Substitution of one or both hydrogen atoms of this group by radicals generates a variety of bisphosphonates; usually they are grouped into two types, namely non-N-BP and N-BP, depending on the absence or presence of a nitrogen atom in its molecule, respectively (Russell, 2011). Examples of BPs used in this report are: etidronate and clodronate (non-N-BPs), and pamidronate and alendronate (N-BPs). Bisphosphonates are the leading drug class for the treatment of osteoporosis; as an indication of their usefulness and the spread out of the disease the combined sales of drugs to treat osteoporosis reached

Novel vaccines targeting dendritic cells by coupling allergoids to nonoxidized mannan enhance allergen uptake and induce functional regulatory T cells through programmed death ligand 1

6.2 billions in 2004. Besides their application in humans, bisphosphonates are also used for other purposes, mainly as herbicides in plants (Cromartie et al., 1999; Oberhauser et al., 1998), as chemotherapeutic agents (Artz et al., 2008; Docampo & Moreno, 2001; Leon et al., 2006; Moreno & Li, 2008) and in basic research (Rogers et al., 2010; Russell, 2011). The usefulness of bisphosphonates is due to their mechanism of action when they are supplied to the living organism. In fact, they have the ability of interfering with metabolic pathways located at the crossroads of essential processes for life. Some important examples of this crucial role are as follows: a) Bisphosphonates may act as analogs of PP in many of the reactions catalyzed by ligases and some transferases (reaction 1) in which derivatives of the type NRpp-CH2-p are synthesized in the reverse reaction (reaction 2) (Gunther Sillero et al., 2008; Gunther Sillero et al., 2006; Rogers et al., 1996; Russell, 2011).